COMPARISON OF GENOMIC 5-METHYLDEOXYCYTIDINE LEVELS IN NEONATAL AND ADULT HUMAN KERATINOCYTES, UNDIFFERENTIATED AND DIFFERENTIATED, IN VITRO

To determine if DNA methylation is a regulator of differentiation in skin, human adult and neonatal keratinocytes, undifferentiated and differentiated, were grown in culture, and genomic methylation was quantified. Key events in the differentiation of keratinocytes are crosslinked envelope formation and the expression of high molecular weight keratins (65-68kD). The latter is in response to new genetic expression, and was the differentiation marker used in this study. Quantitation of methylation was made by separation of radiolabeled nucleotides on HPLC, then scintillation counting fractions corresponding to ultraviolet absorbances of nonradioactive nucleotides contained in the radioactive mix. Methylation levels decreased from 3.2% (undifferentiated average) to 1.2% (differentiated average) in neonatal and adult keratinocytes. Methylations in the undifferentiated keratinocyte genome is about thirty percent higher than in the genome of the differentiated system, leading to the conclusion that methylations do have a function in the differentiation process of keratinocytes in vitro.